Project Summary/Abstract The objective is to produce new classes of near-infrared fluorescent, multivalent molecular probes for advanced cell microscopy studies of membrane receptor clustering and also for paradigm shifting applications in fluorescence guided surgery. There is evidence that multivalent probes bearing multiple copies of a receptor antagonist can trigger receptor clustering and cell endocytosis of the probe, which is highly desired for many imaging applications. But presently it is not possible to produce, by rational design, an effective multivalent molecular probe for reliable in vivo targeting. The first specific aim will utilize a new programmable pre- assembly method to rapidly produce libraries of intrinsically bright, photostable, near-infrared fluorescent molecular probes with systematically altered multivalent binding properties such as ligand loading, linker distance, and degree of PEGylation to control biodistribution. A 35-member library of multivalent probes will be prepared with each member bearing a different copy number of the cyclic pentapeptide, cRGDfK, a targeting ligand for integrin receptors. The fluorescent probes will target a broad range of cancers, but this proposal will focus on epithelial ovarian carcinoma, the most common clinical form of ovarian cancer. The probe library will be screened with ovarian cancer cells for ability to promote extensive integrin-mediated endocytosis. Cell microscopy experiments will use the best probes from the library to visualize and quantify multivalent clustering of integrin receptors, a centrally important but poorly defined process in cell biology events such as signaling, proliferation, migration, and cancer metastasis. The second specific aim will produce two different near-infrared fluorescent multivalent probes for use in fluorescence guided surgery. One sub-aim will produce an in vivo fluorescent probe that can image epithelial ovarian cancer in a clinically relevant mouse model. A novel imaging protocol will be developed to determine if an observed probe signal originates from a tumor nodule on the surface or buried deep in the tissue. The fluorescent probe will enable surgeons to identify tumor nodules that are <1 mm, which is more than ten times smaller than the nodule size currently removed during optimal cytoreductive surgery. Lowering the size of resected ovarian cancer nodules by an order of magnitude is predicted to increase patient survivorship after surgery. A second sub-aim will produce a high performance, fluorescent multivalent peptide probe that can help a surgeon to visualize thin and buried nerves and avoid damaging them. The high overall impact of the proposal derives from the innovation of the versatile pre- assembly synthesis method to rapidly prepare libraries of near-infrared fluorescent multivalent molecular probes for virtually any cell surface biomarker, and the significance of the two proposed fluorescent molecular probes for separate and clinically important applications in fluorescence guided surgery.